Food-borne illness such as food poisoning is a major global threat to health. The Centers for Disease Control and Prevention (CDC) estimates that each year 1 in 6 Americans (about 48 million) people get sick from foodborne pathogenic microbes, about 128,000 are hospitalized, and about 3,000 die of foodborne diseases. In the U.S., about 240,000 illnesses, 1,000 hospitalizations, and 6 deaths per year are thought to be caused by staphylococcal food poisoning. The bacterium, Staphylococcus aureus synthesizes more than two dozen subtypes of staphylococcal enterotoxins (SE) that are responsible for foodborne illness and produces two related but separate biological responses: gastrointestinal targeted emesis and superantigen activation of the immune system. The social and economic impact of such illness is considerable in terms of loss of working days, lower productivity, hospital expenses, and economical losses in the food industry. The most common symptoms caused by ingestion of food contaminated with one or more subtypes of staphylococcal enterotoxins are nausea, violent vomiting, abdominal cramping, and diarrhea. The illness typically resolves within one to two days after onset, but in some cases can be severe enough to require hospitalization. Such toxins are a threat to both food safety and also food security if they are produced in a purified form that can be used as a deliberate adulterant.
One of the most common causes of food poisoning is the bacterium Staphylococcus aureus, which produces a wide range of exotoxins, including many subtypes of staphylococcal enterotoxins that have been associated with significant and frequent outbreaks in many parts of the world, in countries such as the United States, the United Kingdom, Japan, and France (see e.g., Bergdoll M. S., et al., 1971, Identification of enterotoxin E. Infect Immun 4(5):593-5; Wieneke A. A., et al., 1993, Staphylococcal food poisoning in the United Kingdom, 1969-90. Epidemiol Infect 110(3):519-31; Asao T., et al., 2003, An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk. Epidemiol Infect 130(1):33-40; Ostyn A., et al., 2010, First evidence of a food poisoning outbreak due to staphylococcal enterotoxin type E, France, 2009, Euro Surveill 15(13)). Examples of the many sources of potential staphylococcal enterotoxin contamination include manual contact, respiratory secretions, food animals, and dairy cattle. As a result, staphylococcal enterotoxins commonly contaminate meat, poultry, egg, and dairy products as well as vegetables, leafy greens, and baked goods, among others.
Current methods for the detection of active staphylococcal enterotoxin focus on the emetic effect of the toxin and involve the administration of a toxin laden sample to, for example, live monkeys, kittens, or guinea pigs either by gavage or intravenously (see e.g., Fulton, F., 1943, Staphylococcal enterotoxin—with special reference to the kitten test. Brit. J. Exp. Pathol. 24, 65; Bergdoll M. S., et al., 1971, Identification of enterotoxin E. Infect Immun 4(5):593-5; Bergdoll M. S., 1988, Monkey feeding test for staphylococcal enterotoxin. Methods Enzymol 165:324-33; Scheuber, P. H., et al., 1983, Direct skin test in highly sensitized guinea pigs for rapid and sensitive determination of Staphylococcal enterotoxin B. Appl. Environ. Microbiol. 46, 1351-1356). In addition to the ethical and regulatory concerns (e.g., Lautenberg Chemical Safety Act, which promotes the development and use of alternatives to animal testing for chemical toxicity methodologies), these animal model tests are also economically inefficient and have insufficient sensitivity for detecting low quantities of toxin. It has been reported that the animal-based method is not very sensitive and generally requires 5 to 20 mg of SE to cause 50% emesis in young monkeys (see e.g., Su, Y C & Wong, A C, Identification and Purification of a New Staphylococcal Enterotoxin, H., Appl Environ Microbiol, 1995, April 61(4), 1438-4343). In another report, it was shown that the response of monkeys to staphylococcal enterotoxins depends on the method of administration. For example, it was found that for intragastric administration the level of detection was 10 μg and for intravenous injection it was 0.5 μg (see e.g., Bergdoll M S, et al, Identification of enterotoxin E, Infect Immun, 1971, 4(5):593-5). Much lower limits of detection without the use of animal models are needed for industrial use and efficiency.
An alternative method to detect microbial toxins is chemically via, for example, enzyme-linked immunosorbent assay (ELISA) or by mass spectrometry (MS) (see e.g., Bennett R. W., 2005. Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology. J Food Prot 68(6):1264-70; Dupuis A. et al., 2008, Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks. Proteomics 8(22):4633-6). The lower limit of detection with such methods is generally about 0.25 to 1.0 ng/gr food (see e.g., Bennett R. W., 2005. Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology. J Food Prot 68(6):1264-70). The main drawbacks of these chemical methods is the requirement of expensive instrumentation that is generally beyond the means of laboratories without many resources, and, in addition, the assays are unable to discern active toxin from inactivated toxin. Discerning between active and inactive toxin is important because food that has been treated or processed to reduce or eliminate toxin by inactivation may still test positive with these non-mechanistic (i.e., unable to discern between active and inactive toxin) assays.
There thus exists a challenging and urgent need for new systems and methods of detecting and identifying microbial toxins in a rapid, accurate, sensitive, and cost-effective fashion for governmental and non-governmental agencies, including the military, public health departments, healthcare facilities, and the food industry.